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1.
Int J Ophthalmol ; 14(12): 1915-1920, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34926208

RESUMO

AIM: To quantify the area and density of retinal vascularity by ultra-widefield fluorescein angiography (UWFA). METHODS: In a retrospective study, UWFA images were obtained using an ultra-widefield imaging device in 42 normal eyes of 42 patients. Central and peripheral steered images were used to define the edge of retinal vasculature by a certified grader. The length from the center of the optic disc to the edge of retinal vascularity (RVL) in each quadrant and the total retinal vascular perfusion area (RVPA) were determined by the grader using OptosAdvance software. The density of retinal vascularity (RVD) was quantified in different zones of central-steered images using Image J software. RESULTS: Among 42 healthy eyes, the values for mean RVL in each quadrant were 19.007±0.781 mm (superior), 18.467±0.869 mm (inferior), 17.738±0.622 mm (nasal) and 24.241±1.336 mm (temporal). The mean RVPA was 1140.117±73.825 mm2. The mean RVD of the total retina was 4.850%±0.638%. RVD varied significantly between different retina zones (P<0.001), and significant differences existed in the RVD values for total retinal area in patients over 50 years old compared to those under 50 years old (P=0.033). No gender difference was found. CONCLUSION: The UWFA device can be a promising tool for analyzing the overall retinal vasculature and may provide a better understanding of retinal vascular morphology in normal eyes. Aging may be related to lower RVD.

2.
PeerJ ; 8: e9397, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32587808

RESUMO

BACKGROUND: Previous studies recruited unrepresentative samples of Chinese patients with cataract and reported a wide range of prevalence of depressive symptoms in this patient population (18.0-89.7%). The present study determined the prevalence and correlates of depressive symptoms among a consecutive sample of Chinese patients with cataract treated in tertiary general hospitals. METHODS: A total of 339 patients with cataract were consecutively selected from ophthalmology departments of two large general hospitals in Wuhan, China. Depressive symptoms were assessed with the Chinese Hospital Anxiety and Depression Scale. Logistic regression was used to identify factors that were associated with depression. RESULTS: The prevalence of depressive symptoms was 23.9% (95% CI [19.4-28.4]%) among patients with cataract. Correlates for depressive symptoms include an education level of primary school and below (OR = 1.93, P = 0.038), marital status of "others" (OR =3.15, P < 0.001), poor family economic status (OR = 2.26, P = 0.010), nuclear cataract (OR =4.32, P < 0.001), and mixed cataract (OR = 2.76, P = 0.017). CONCLUSIONS: Depressive symptoms are common among Chinese patients with cataract treated in large general hospitals. Patients who are poorly educated, have a marital status other than "married", have poor family economic status, and suffer from nuclear and mixed cataracts are at greater risk for depressive symptoms.

3.
J Clin Invest ; 130(4): 1879-1895, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31874105

RESUMO

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.


Assuntos
Carcinogênese/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Família Multigênica , Neoplasias Experimentais/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Neoplásico/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Neoplasias Experimentais/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Neoplásico/genética
4.
Int J Ophthalmol ; 10(4): 524-529, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28503422

RESUMO

AIM: To investigate the effect of HtrA1 on the proliferation, migration and apoptosis of human retinal pigment epithelium (RPE) cells in the light injured model, as well as the expression of the apoptosis related molecules. METHODS: The human RPE cell line ARPE-19 was exposed to blue light to establish the light injured model. The cells were transfected with HtrA1 siRNA to knockdown HtrA1 expression. Subsequent expression of HtrA1 was determined by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. Changes in cell proliferation, migration and apoptosis were assessed by cell counting kit-8 (CCK-8), Transwell assay and flow cytometry respectively, as well as changes in the mRNA and protein levels of Bax, Caspase-3 and Bcl-2 expression. RESULTS: HtrA1 was highly expressed in ARPE-19 cells after blue light irradiation. Knockdown of HtrA1 expression inhibited the proliferation, migration and apoptosis of the blue-light-irradiated ARPE-19 cells (P<0.05). Bax and Caspase-3 expression were significantly reduced both at mRNA and protein levels (P<0.05) after siRNA treatment. Bcl-2 expression significantly increased in blue-light-irradiated ARPE-19 cells after siRNA interference (P<0.05). CONCLUSION: Silence of HtrA1 may inhibit the proliferation, migration and apoptosis of ARPE-19 cells in light injured model. Moreover, HtrA1 suppression in blue-light-irradiated ARPE-19 cells may ameliorate cell apoptosis through down-regulation of Bax and Caspase-3, and up-regulation of Bcl-2 expression.

5.
J Immunol ; 195(4): 1470-9, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26163591

RESUMO

Understanding the consequences of tuning TCR signaling on selection, peripheral T cell function, and tolerance in the context of native TCR repertoires may provide insight into the physiological control of tolerance. In this study, we show that genetic ablation of a natural tuner of TCR signaling, mir-181a-1/b-1, in double-positive thymocytes dampened TCR and Erk signaling and increased the threshold of positive selection. Whereas mir-181a-1/b-1 deletion in mice resulted in an increase in the intrinsic reactivity of naive T cells to self-antigens, it did not cause spontaneous autoimmunity. Loss of mir-181a-1/b-1 dampened the induction of experimental autoimmune encephalomyelitis and reduced basal TCR signaling in peripheral T cells and their migration from lymph nodes to pathogenic sites. Taken together, these results demonstrate that tolerance can be modulated by microRNA gene products through the control of opposing activities in T cell selection and peripheral T cell function.


Assuntos
Seleção Clonal Mediada por Antígeno/genética , Seleção Clonal Mediada por Antígeno/imunologia , Tolerância Imunológica/genética , MicroRNAs/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Autoimunidade , Movimento Celular/genética , Movimento Celular/imunologia , Modelos Animais de Doenças , Fosfatase 6 de Especificidade Dupla/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Deleção de Genes , Imunização , Lisofosfolipídeos/imunologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Oligonucleotídeos/genética , Interferência de RNA , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/imunologia , Timócitos/imunologia , Timócitos/metabolismo
6.
PLoS Genet ; 11(2): e1005020, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25714103

RESUMO

The development and homeostasis of multicellular organisms relies on gene regulation within individual constituent cells. Gene regulatory circuits that increase the robustness of gene expression frequently incorporate microRNAs as post-transcriptional regulators. Computational approaches, synthetic gene circuits and observations in model organisms predict that the co-regulation of microRNAs and their target mRNAs can reduce cell-to-cell variability in the expression of target genes. However, whether microRNAs directly regulate variability of endogenous gene expression remains to be tested in mammalian cells. Here we use quantitative flow cytometry to show that microRNAs impact on cell-to-cell variability of protein expression in developing mouse thymocytes. We find two distinct mechanisms that control variation in the activation-induced expression of the microRNA target CD69. First, the expression of miR-17 and miR-20a, two members of the miR-17-92 cluster, is co-regulated with the target mRNA Cd69 to form an activation-induced incoherent feed-forward loop. Another microRNA, miR-181a, acts at least in part upstream of the target mRNA Cd69 to modulate cellular responses to activation. The ability of microRNAs to render gene expression more uniform across mammalian cell populations may be important for normal development and for disease.


Assuntos
Sobrevivência Celular/genética , MicroRNAs/genética , Biossíntese de Proteínas/genética , Timócitos/metabolismo , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Camundongos , RNA Mensageiro/biossíntese
7.
Biotechniques ; 57(3): 115-24, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25209046

RESUMO

The prokaryotic type II CRISPR/Cas9 system has been adapted to perform targeted genome editing in cells and model organisms. Here, we describe targeted gene deletion and replacement in human cells via the CRISPR/Cas9 system using two guide RNAs. The system effectively generated targeted deletions of varied length, regardless of the transcriptional status of the target gene. It is notable that targeted gene deletions generated via CRISPR/Cas9 and two guide RNAs resulted in the formation of correct junctions at high efficiency. Moreover, in the presence of a homology repair donor, the CRISPR/Cas9 system could guide precise gene replacement. Our results illustrate that the CRISPR/Cas9 system can be used to precisely and effectively generate targeted deletions or gene replacement in human cells, which will facilitate characterization of functional domains in protein-coding genes as well as noncoding regulatory sequences in animal genomes.


Assuntos
Sistemas CRISPR-Cas/genética , Deleção de Genes , Técnicas Genéticas , Células HEK293 , Humanos
8.
Nat Med ; 20(7): 769-77, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24859528

RESUMO

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.


Assuntos
Transformação Celular Neoplásica/genética , Trato Gastrointestinal/patologia , Oncogenes , Animais , Neoplasias Gastrointestinais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos
9.
Int J Ophthalmol ; 7(1): 8-13, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24644534

RESUMO

AIM: To investigate the protective mechanism of Gingko Biloba extract (EGb761) on the ability of retinal pigment epithelial (RPE) cells to resist light-induced damage in a comparative proteomics study. METHODS: Human RPE cells (ARPE-19) were randomly distributed to one of three groups: normal control (NC group) and light-damaged model without or with EGb761 group (M and ME groups, respectively). The light-damaged model was formed by exposing to white light (2 200±300)lx for 6h. The RPE cells in ME group were conducted with EGb 761 (100µg/mL) before light exposure. The soluble cellular proteins extracting from each groups were separated by two-dimensional electrophoresis and stained by silver staining. Different proteins in the profiles of the gels were analyzed by Image Master Software. Two-fold expressing protein spots were identified by Matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. RESULTS: NC, M and ME groups displayed 1 892±71, 2 145±23 and 2 216±85 protein spots, respectively. We identified 33 proteins with different expression levels between the NC and M groups, 25 proteins between the M and ME groups, and 11 proteins between the NC and ME groups. MALDI-TOF/TOF mass spectrometry successfully identified 16 proteins, including metabolic enzymes, cytoskeletal proteins, anti-oxidation proteins, and others. CONCLUSION: Differences in some important proteins, such as cathepsin B, heat shock protein, and cytochrome c reductase, indicated that multiple pathways may be induced in light-damaged RPE cells and the protective effect of EGb761.

10.
Biochimie ; 95(9): 1663-76, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23685275

RESUMO

MicroRNAs (miRNAs) are an abundant class of endogenous ∼21-nucleotide (nt) RNAs. These small RNAs are produced from long primary miRNA transcripts - pri-miRNAs - through sequential endonucleolytic maturation steps that yield precursor miRNA (pre-miRNA) intermediates and then the mature miRNAs. The mature miRNAs are loaded into the RNA-induced silencing complexes (RISC), and guide RISC to target mRNAs for cleavage and/or translational repression. This paradigm, which represents one of major discoveries of modern molecular biology, is built on the assumption that mature miRNAs are the only species produced from miRNA genes that recognize targets. This assumption has guided the miRNA field for more than a decade and has led to our current understanding of the mechanisms of target recognition and repression by miRNAs. Although progress has been made, fundamental questions remain unanswered with regard to the principles of target recognition and mechanisms of repression. Here I raise questions about the assumption that mature miRNAs are the only target-recognizing species produced from miRNA genes and discuss the consequences of working under an incomplete or incorrect assumption. Moreover, I present evolution-based and experimental evidence that support the roles of pri-/pre-miRNAs in target recognition and repression. Finally, I propose a conceptual framework that integrates the functions of pri-/pre-miRNAs and mature miRNAs in target recognition and repression. The integrated framework opens experimental enquiry and permits interpretation of fundamental problems that have so far been precluded.


Assuntos
MicroRNAs/genética , Animais , Regulação da Expressão Gênica , Humanos , Precursores de RNA/genética , RNA Antissenso/genética , RNA Bacteriano/genética
11.
Immunol Rev ; 253(1): 112-28, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23550642

RESUMO

Much has been learned about the molecular and cellular components critical for the control of immune responses and tolerance. It remains a challenge, however, to control the immune response and tolerance at the system level without causing significant toxicity to normal tissues. Recent studies suggest that microRNA (miRNA) genes, an abundant class of non-coding RNA genes that produce characteristic approximately 22 nucleotides small RNAs, play important roles in immune cells. In this article, we discuss emerging knowledge regarding the functions of miRNA genes in the immune system. We delve into the roles of miRNAs in regulating signaling strength and threshold, homeostasis, and the dynamics of the immune response and tolerance during normal and pathogenic immunological conditions. We also present observations based on analyzes of miR-181 family genes that indicate the potential functions of primary and/or precursor miRNAs in target recognition and explore the impact of these findings on target identification. Finally, we illustrate that despite the subtle effects of miRNAs on gene expression, miRNAs have the potential to influence the outcomes of normal and pathogenic immune responses by controlling the quantitative and dynamic aspects of immune responses. Tuning miRNA functions in immune cells, through gain- and loss-of-function approaches in mice, may reveal novel approach to restore immune equilibrium from pathogenic conditions, such as autoimmune disease and leukemia, without significant toxicity.


Assuntos
Tolerância Imunológica , Imunidade , Imunomodulação , MicroRNAs/imunologia , Animais , Homeostase/genética , Homeostase/imunologia , Humanos , Tolerância Imunológica/genética , Imunidade/genética , Camundongos , Transdução de Sinais/genética , Transdução de Sinais/imunologia
12.
PLoS Genet ; 8(8): e1002855, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22916024

RESUMO

Oncogenes, which are essential for tumor initiation, development, and maintenance, are valuable targets for cancer therapy. However, it remains a challenge to effectively inhibit oncogene activity by targeting their downstream pathways without causing significant toxicity to normal tissues. Here we show that deletion of mir-181a-1/b-1 expression inhibits the development of Notch1 oncogene-induced T cell acute lymphoblastic leukemia (T-ALL). mir-181a-1/b-1 controls the strength and threshold of Notch activity in tumorigenesis in part by dampening multiple negative feedback regulators downstream of NOTCH and pre-T cell receptor (TCR) signaling pathways. Importantly, although Notch oncogenes utilize normal thymic progenitor cell genetic programs for tumor transformation, comparative analyses of mir-181a-1/b-1 function in normal thymocyte and tumor development demonstrate that mir-181a-1/b-1 can be specifically targeted to inhibit tumor development with little toxicity to normal development. Finally, we demonstrate that mir-181a-1/b-1, but not mir-181a-2b-2 and mir-181-c/d, controls the development of normal thymic T cells and leukemia cells. Together, these results illustrate that NOTCH oncogene activity in tumor development can be selectively inhibited by targeting the molecular networks controlled by mir-181a-1/b-1.


Assuntos
Regulação Leucêmica da Expressão Gênica , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores Notch/genética , Transdução de Sinais/genética , Linfócitos T/metabolismo , Animais , Transformação Celular Neoplásica/genética , Humanos , Camundongos , MicroRNAs/metabolismo , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismo , Linfócitos T/patologia , Timo/metabolismo , Timo/patologia
13.
RNA Biol ; 8(6): 1115-23, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22142974

RESUMO

MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5' end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs' sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation.


Assuntos
MicroRNAs/genética , Nucleotídeos/genética , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , Animais , Sequência de Bases , Northern Blotting , Caenorhabditis elegans/genética , Linhagem Celular , Regulação da Expressão Gênica , Humanos , MicroRNAs/química , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , Precursores de RNA/química , Precursores de RNA/metabolismo
14.
Ophthalmologica ; 226(4): 220-7, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21811053

RESUMO

PURPOSE: To study the clinical usage of sweep pattern visual evoked potential (SPVEP) acuity in children's visual development periods and compare the amplitude-spatial frequency (A-SP) function regression method with the amplitude-logarithm of the visual angle (A-logVA) function regression method in evaluating the SPVEP acuity of children, especially those who have poor visual acuities. METHODS: Twenty-six eyes of 26 amblyopic children (ages ranged from 3 to 12 years; mean age±standard deviation 6.69±1.74 years) and 31 eyes of normal children whose ages were matched with the amblyopic group were involved in this study. SPVEP acuity was recorded with GT-2000 NV (Guote Medical Apparatus Ltd., China) using sinusoidally modulated horizontal gratings with 10 different spatial frequencies from 0.99 to 12.89 cycles per degree to stimulate the retina. The averaging responses were displayed with the discrete Fourier transformation method. SPVEP acuity was assessed by both the A-SP function regression method and the A-logVA function regression method. The logarithm of minimal angle of resolution (logMAR) chart was used to obtain logMAR visual acuity. RESULTS: In the normal group, logMAR acuity calculated by both the A-SP and A-logVA function regression methods had a significant correlation with SPVEP acuity. The average value of SPVEP acuity (by A-logVA) was closer to logMAR acuity. The difference of mean values between logMAR acuity and SPVEP acuity was significant in both regression methods. In the amblyopic group, it was SPVEP acuity (by A-logVA) that had a significant correlation with logMAR acuity, whereas the result was not significant when calculated by the A-SP function regression method (p=0.515). The average value of SPVEP acuity (A-SP) was closer to logMAR acuity. The difference of mean values between logMAR acuity and SPVEP acuity (A-logVA) was significant; however, when compared with SPVEP acuity (A-SP), it was not significant (p=0.174). In addition, SPVEP acuity may be overestimated or underestimated when it is compared with different logMAR visual acuities. CONCLUSION: SPVEP could be used to evaluate the visual acuity for normal children or those with poor visual acuity. Moreover, the A-logVA function regression method was more accurate than the A-SP function regression method in evaluating SPVEP acuity.


Assuntos
Potenciais Evocados Visuais/fisiologia , Olho/crescimento & desenvolvimento , Transtornos da Visão/diagnóstico , Acuidade Visual/fisiologia , Córtex Visual/fisiologia , Ambliopia/fisiopatologia , Criança , Pré-Escolar , Feminino , Análise de Fourier , Humanos , Masculino , Erros de Refração/fisiopatologia , Estrabismo/fisiopatologia , Transtornos da Visão/fisiopatologia
15.
Genome Res ; 21(5): 798-810, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21451113

RESUMO

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/genética , MicroRNAs/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Células-Tronco Neurais , Especificidade de Órgãos , Células-Tronco/citologia
16.
EMBO J ; 29(19): 3272-85, 2010 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-20808284

RESUMO

Major RNA products of a microRNA (miRNA) gene--the long primary transcript (pri-miRNA), the ∼70-nucleotide (nt) precursor miRNA (pre-miRNA), and the ∼21-nt mature miRNA--all contain the same sequence required for target gene recognition. Thus, it is intrinsically difficult to discern the contribution of individual RNA species or to rule out a function of miRNA precursor species in target repression. Here, we describe a novel approach to dissect the functional contribution of pri-miRNA without compromising important cellular pathways. We show that pri-let-7 has a direct function in target repression in the absence of properly processed mature let-7. Moreover, we show that loop nucleotides provide regulatory controls of the activity of pri-let-7 by modulating interactions between pri-let-7 and target RNAs in vitro and in vivo. Finally, we show that human let-7a-3 pri-miRNA can directly interact with target mRNAs. These findings illustrate that the regulatory information encoded in structured pri-miRNAs may be translated into function through direct interactions with target mRNAs.


Assuntos
Regulação da Expressão Gênica/fisiologia , MicroRNAs/fisiologia , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Northern Blotting , Caenorhabditis elegans , Linhagem Celular , Regulação da Expressão Gênica/genética , Humanos , Camundongos , MicroRNAs/biossíntese , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação/genética , Reação em Cadeia da Polimerase
17.
Cancer Res ; 70(9): 3833-42, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20406979

RESUMO

Despite advances in defining the critical molecular determinants for leukemia stem cell (LSC) generation and maintenance, little is known about the roles of microRNAs in LSC biology. Here, we identify microRNAs that are differentially expressed in LSC-enriched cell fractions (c-kit(+)) in a mouse model of MLL leukemia. Members of the miR-17 family were notably more abundant in LSCs compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Expression of miR-17 family microRNAs was substantially reduced concomitant with leukemia cell differentiation and loss of self-renewal, whereas forced expression of a polycistron construct encoding miR-17-19b miRNAs significantly shortened the latency for MLL leukemia development. Leukemias expressing increased levels of the miR-17-19b construct displayed a higher frequency of LSCs, more stringent block of differentiation, and enhanced proliferation associated with reduced expression of p21, a cyclin-dependent kinase inhibitor previously implicated as a direct target of miR-17 microRNAs. Knockdown of p21 in MLL-transformed cells phenocopied the overexpression of the miR-17 polycistron, including a significant decrease in leukemia latency, validating p21 as a biologically relevant and direct in vivo target of the miR-17 polycistron in MLL leukemia. Expression of c-myc, a crucial upstream regulator of the miR-17 polycistron, correlated with miR-17-92 levels, enhanced self-renewal, and LSC potential. Thus, microRNAs quantitatively regulate LSC self-renewal in MLL-associated leukemia in part by modulating the expression of p21, a known regulator of normal stem cell function.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , MicroRNAs/genética , Células Progenitoras Mieloides/patologia , Células-Tronco Neoplásicas/patologia , Animais , Ciclo Celular/genética , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes , Genes myc , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/biossíntese , Proteína de Leucina Linfoide-Mieloide/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Pathobiology ; 77(2): 88-95, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20332668

RESUMO

Proliferation and migration of retinal pigment epithelial (RPE) cells play a crucial role in proliferative vitreoretinopathy (PVR)-related pathology. Cytokines, including EGF, can result in RPE cell activation and cause PVR. In this study, integrin-alpha(5) expression was first studied in PVR membranes by immunofluorescence. Then the effect of EGF on integrin-alpha(5) expression was determined by flow cytometry, Western blot analysis and the reverse-transcription polymerase chain reaction (RT-PCR) in the ARPE-19 cell line. Proliferation and migration of ARPE-19 cells were measured by the methylthiazolyldiphenyl-tetrazolium bromide and Boyden chamber assays. We found that a higher level integrin-alpha(5) was present at the RPE cell surface in PVR compared with normal retina. EGF could dose dependently increase integrin-alpha(5) mRNA and protein levels in vitro. EGF promoted ARPE-19 cell proliferation and migration. Neutralizing integrin-alpha(5) by specific anti-integrin-alpha(5) antibody abolished most of the effects of EGF. The study provided evidence that EGF might influence PVR by promoting integrin-alpha(5) expression and subsequent proliferation and migration of RPE.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Integrina alfa5/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Vitreorretinopatia Proliferativa/metabolismo , Adulto , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfa5/genética , Masculino , Pessoa de Meia-Idade , Vitreorretinopatia Proliferativa/patologia
19.
Immunity ; 30(5): 744-52, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19446474

RESUMO

The full set of microRNAs (miRNAs) in the human genome is not known. Because presently known miRNAs have been identified by virtue of their abundant expression in a few cell types, many tissue-specific miRNAs remain unrevealed. To understand the role of miRNAs in B cell function and lymphomagenesis, we generated short-RNA libraries from normal human B cells at different stages of development (naive, germinal center, memory) and from a Burkitt lymphoma cell line. A combination of cloning and computational analysis identified 178 miRNAs (miRNome) expressed in normal and/or transformed B cell libraries. Most notably, the B cell miRNome included 75 miRNAs which to our knowledge have not been previously reported and of which 66 have been validated by RNA blot and/or RT-PCR analyses. Numerous miRNAs were expressed in a stage- or transformation-specific fashion in B cells, suggesting specific functional or pathologic roles. These results provide a resource for studying the role of miRNAs in B cell development, immune function, and lymphomagenesis.


Assuntos
Subpopulações de Linfócitos B/imunologia , MicroRNAs/fisiologia , Animais , Linhagem Celular Tumoral , Cães , Evolução Molecular , Regulação da Expressão Gênica , Haplorrinos , Humanos , Camundongos , MicroRNAs/análise , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Tonsila Palatina/metabolismo , Ratos
20.
PLoS One ; 3(10): e3592, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18974849

RESUMO

BACKGROUND: Mature miRNAs can often be classified into large families, consisting of members with identical seeds (nucleotides 2 through 7 of the mature miRNAs) and highly homologous approximately 21-nucleotide (nt) mature miRNA sequences. However, it is unclear whether members of a miRNA gene family, which encode identical or nearly identical mature miRNAs, are functionally interchangeable in vivo. METHODS AND FINDINGS: We show that mir-181a-1, but not mir-181c, can promote CD4 and CD8 double-positive (DP) T cell development when ectopically expressed in thymic progenitor cells. The distinct activities of mir-181a-1 and mir-181c are largely determined by their unique pre-miRNA loop nucleotides-not by the one-nucleotide difference in their mature miRNA sequences. Moreover, the activity of mir-181a-1 on DP cell development can be quantitatively influenced by nucleotide changes in its pre-miRNA loop region. We find that both the strength and the functional specificity of miRNA genes can be controlled by the pre-miRNA loop nucleotides. Intriguingly, we note that mutations in the pre-miRNA loop regions affect pre-miRNA and mature miRNA processing, but find no consistent correlation between the effects of pre-miRNA loop mutations on the levels of mature miRNAs and the activities of the mir-181a-1/c genes. CONCLUSIONS: These results demonstrate that pre-miRNA loop nucleotides play a critical role in controlling the activity of miRNA genes and that members of the same miRNA gene families could have evolved to achieve different activities via alterations in their pre-miRNA loop sequences, while maintaining identical or nearly identical mature miRNA sequences.


Assuntos
Diferenciação Celular/genética , MicroRNAs/fisiologia , Conformação de Ácido Nucleico , Linfócitos T/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Hematopoese/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/química , Modelos Biológicos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Linfócitos T/metabolismo
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